Interferons are classified either as the leukocyte and fibroblast derived Type I interferons, or as the mitogen induced or “immune” Type II interferons (Pestka et al, 1987). Through analysis of sequence identities and common biological activities, type I interferons include interferon alpha (IFNα), interferon beta (IFNβ) and interferon omega (IFNω), while type II interferon includes interferon gamma (IFNγ).
The IFNα, IFNβ and IFNω genes are clustered on the short arm 25 of chromosome 9 (Lengyl, 1982). There are at least 25 non-allelic IFNα genes, 6 non-allelic IFNω genes and a single IFNβ gene. All are believed to have evolved from a single common ancestral gene. Within species, IFNα genes share at least 80% sequence identity with each other. The IFNβ gene shares approximately 50% sequence identity with IFNα; and the IFNω gene shares 70% homology with IFNα (Weissman et al, 1986; Dron et al, 1992). IFNα has a molecular weight range of 17-23 kDa (165-166 amino acids), IFNβ, about 23 kDa (166 amino acids) and IFNω, about 24 kDa (172 amino acids).
Type I interferons are pleiotropic cytokines having activity such as host defense against viral and parasitic infections, anti-cancer properties and as immune modulators (Baron et al, 1994; Baron et al, 1991). Type I interferon physiological responses include anti-proliferative activity on normal and transformed cells, stimulation of cytotoxic activity in lymphocytes, natural killer cells and phagocytic cells, modulation of cellular differentiation, stimulation of expression of class I MHC antigens, inhibition of class II MHC, and modulation of a variety of cell surface receptors. Under normal physiological conditions, IFNα and IFNβ (IFNα/β) are secreted constitutively by most human cells at low levels with expression being up-regulated by addition of a variety of inducers, comprising infectious agents (viruses, bacteria, mycoplasma and protozoa), dsRNA, and cytokines (M-CSF, IL-1α, IL-2, TNFα). The actions of Type I interferon in vivo can be monitored using the surrogate markers, neopterin, 2′,5′ oligoadenylate synthetase, and β2 microglobulin (Alam et al, 1997; Fierlbeck et al, 1996; Salmon et al, 1996).
Type I interferons (IFNα/β/ω) act through a cell surface receptor complex to induce specific biologic effects, such as anti-viral, anti-tumor, and immune modulators. The type I IFN receptor (IFNAR) is a hetero-multimeric receptor complex composed of at least two different polypeptide chains (Colamonici et al, 1992; Colamonici et al, 1993; Platanias et al, 1993). The genes coding for these chains are found on chromosome 21, and their proteins are expressed on the surface of most cells (Tan et al, 1973). The receptor chains were originally designated alpha and beta and have been renamed IFNAR1 for the alpha subunit and IFNAR2 for the beta subunit. In most cells, IFNAR1 (alpha chain, Uze subunit) (Uze et al, 1990) has a molecular weight of 100-130 kDa, while IFNAR2 (beta chain, βL, IFNα/βR) has a molecular weight of 100 kDa. In certain cell types (monocytic cell lines and normal bone marrow cells) an alternate receptor complex has been identified, where the IFNAR2 subunit (βS) is expressed as a truncated receptor with a molecular weight of 51 kDa. The IFNAR1 and IFNAR2 βS and βL subunits have been cloned (Novick et al, 1994; Domanski et al, 1995). The IFNAR2 βS and βL subunits have identical extracellular and transmembrane domains; however, in the cytoplasmic domain they only share identity in the first 15 amino acids. The IFNAR2 subunit alone is able to bind IFNα/β, while the IFNAR1 subunit is unable to bind IFNα/β. When the human IFNAR1 receptor subunit alone was transfected into murine L-929 fibroblasts, no human IFNαs except IFNα8/IFNαB were able to bind to the cells (Uze et al, 1990). The human IFNAR2 subunit, transfected into L cells in the absence of the human IFNAR1 subunit, bind human IFNα, binding with a Kd of approximately 0.45 nM. When human IFNAR2 subunits were transfected in the presence of the human IFNAR1 subunit, high affinity binding could be shown with a Kd of 0.026-0.114 nM (Novick et al, 1994; Domanski et al, 1995). It is estimated that from 500-20,000 high affinity and 2,000-100,000 low affinity IFN binding sites exist on most cells. Although the IFNAR1/2 complex (α/βs or α/βL) subunits bind IFNα with high affinity, only the α/βL pair appears to be a functional signaling receptor.
Transfection of the IFNAR1 and the IFNAR2 βL subunits into mouse L-929 cells, followed by incubation with IFNα2, induces an anti-viral state, initiates intracellular protein phosphorylation, and causes the activation of intracellular kinases (Jak1 and Tyk2) and transcription factors (STAT 1, 2, and 3) (Novick et al, 1994; Domanski et al, 1995). In a corresponding experiment, transfection of the IFNAR2 βs subunit was unable to initiate a similar response. Thus, the IFNAR2 βL subunit is required for functional activity (anti-viral response) with maximal induction occurring in association with the IFNAR1 subunit.
In addition to membrane bound cell surface IFNAR forms, a soluble IFNAR has been identified in both human urine and serum (Novick et al, 1994; Novick et al, 1995; Novick et al, 1992; Lutfalla et al, 1995). The soluble IFNAR isolated from serum has an apparent molecular weight of 55 kDa on SDS-PAGE, while the soluble IFNAR from urine has an apparent molecular weight of 40-45 kDa (p40). Transcripts for the soluble p40 IFNAR2 are present at the mRNA level and encompass almost the entire extracellular domain of the IFNAR2 subunit with two additional amino acids at the carboxy terminal end. There are five potential glycosylation sites on the soluble IFNAR2 receptor. The soluble p40 IFNAR2 has been shown to bind IFNα2 and IFNβ and to inhibit in vitro the anti-viral activity of a mixture of IFNα species (“leukocyte IFN”) and individual Type I IFNs (Novick et al, 1995). A recombinant IFNAR2 subunit Ig fusion protein was shown to inhibit the binding of a variety of Type I IFN species (IFNαA, IFNαB, IFNαD, IFNβ, IFNαCon1 and IFNω) to Daudi cells and α/βS subunit double transfected COS cells.
Type I IFN signaling pathways have been identified (Platanias et al, 1996; Yan et al., 1996; Qureshi et al., 1996; Duncan et al., 1996; Sharf et al, 1995; Yang et al, 1996). Initial events leading to signaling are thought to occur by the binding of IFNα/β/ω to the IFNAR2 subunit, followed by the IFNAR1 subunit associating to form an IFNAR1/2 complex (Platanias et al., 1994). The binding of IFNα/β/ω to the IFNAR1/2 complex results in the activation of two Janus kinases (Jak1 and Tyk2), which are believed to phosphorylate specific tyrosines on the IFNAR1 and IFNAR2 subunits. Once these subunits are phosphorylated, STAT molecules (STAT 1, 2 and 3) are phosphorylated, which results in dimerization of STAT transcription complexes followed by nuclear localization of the transcription complex and the activation of specific IFN inducible genes.
A randomized, double-blinded, placebo-controlled, two-year multicenter study demonstrated that natural human fibroblast interferon (interferon beta) administered intrathecally (IT) is effective in reducing the exacerbations of exacerbating-remitting multiple sclerosis (MS). The mean reduction in exacerbation rate of 34 patients with MS who received interferon beta administered IT was significantly greater during the study than that of 35 control patients who received placebo (Jacobs et al. 1987).
The pharmacokinetics and pharmacodynamics of Type I IFNs have been assessed in humans (Alan et al, 1997; Fierlbeck et al, 1996; Salmon et al, 1996). The clearance of IFNβ is fairly rapid with the bioavailability of IFNβ lower than expected for most cytokines. Although the pharmacodynamics of IFNβ has been assessed in humans, no clear correlation has been established between the bioavailability of IFNβ and clinical efficacy. In normal healthy human volunteers, administration of a single intravenous (iv) bolus dose (6 MIU) of recombinant CHO derived IFNβ resulted in a rapid distribution phase of 5 minutes and a terminal half-life of about 5 hours (Alam et al, 1997). Following subcutaneous (sc) or intramuscular (im) administration of IFNβ, serum levels are flat with only about 15% of the dose systemically available. The pharmacodynamics of IFNβ following iv, im or sc administration (as measured by changes in 2′5/-oligoadenylate synthetase (2′,5′-AS) activity in PBMCs) were elevated within the first 24 hours and slowly decreased to baseline levels over the next 4 days. The magnitude and duration of the biologic effect was the same regardless of the route of administration.
A multiple dose pharmacodynamic study of IFNβ has been conducted in human melanoma patients (Fierlbeck et al, 1996) with IFNβ being administrated by sc route, three times per week at 3 MIU/dose over a six-month period. The pharmacodynamic markers, 2′,5′-AS synthetase, β2-microglobulin, neopterin, and NK cell activation peaked by the second injection (day 4) and dropped off by 28 days, remaining only slightly elevated out to six months.
Purification and refolding of the extracellular part of human IFNAR2 (IFNAR2-EC) expressed in Escherichia coli and its characterization with respect to its interaction with interferon alpha2 (IFNα2) has been reported (Piehler and Schreiber 199A). The 25 kDa, non-glycosylated IFNAR2-EC was shown to be a stable, fully active protein, which inhibits antiviral activity of IFNα2. The stoichiometry of binding IFNα2 is 1:1, as determined by gel filtration, chemical cross-linking and solid-phase detection. The affinity of this interaction was found to be about 3 nM (Piehler and Schreiber 2001). The rate of complex formation is relatively high compared to other cytokine-receptor interactions. The salt dependence of the association kinetics suggests a limited but significant contribution of electrostatic forces towards the rate of complex formation. The dissociation constant increases with decreasing pH according to the protonation of a base with a pKa of 6.7. The affinity of IFNβ to IFNAR2 is about two-fold higher than that of IFNα2 to IFNAR2 (Piehler and Schreiber 1999B).
Single mutations in the binding site of IFNAR2 allowed mapping of differences in binding of IFα2 and IFNβ (Piehler and Schreiber 1999B). For example, a mutation H78A was found to stabilize the complex with IFNβ nearly by two fold, while destabilized the complex with IFα2 more than two fold. A mutation N100A was found to hardly affect the rates for binding IFα2, whereas it decreased the dissociation rate constant for IFNβ by almost four fold.
EP1037658 discloses that the in vivo effect of Type I interferon (IFN) can be prolonged by administering the interferon in the form of a complex with an IFN binding chain of the human interferon alpha/beta receptor (IFNAR) i.e. IFNAR behaves as a carrier protein for IFN. Such a complex also improves the stability of the IFN and enhances the potency of the IFN. The complex may be a non-covalent complex or one in which the IFN and the IFNAR are bound by a covalent bond or a peptide. EP1037658 also discloses that storing IFN in the form of such a complex improves the storage life of the IFN and permits storage under milder conditions than would otherwise be possible.
There exists a need for an IFNAR2 with improved affinity towards IFNβ, but not to IFNα2, making IFNAR2 a better and specific carrier for IFNβ.